The principal investigator proposes to use Vibrio cholerae, the causative agent of cholera, as a live oral attenuated vaccine vector to deliver immunogenic antigens of enterohemmorrhagic Escherichia coli (EHEC) to the intestine to stimulate a common mucosal immune response. EHEC, a bacterium that causes hemorrhagic colitis and hemolytic uremic syndrome, is an important pathogen of humans which, like V, cholerae, acts by adhering to the mucosal surface of the intestine and producing toxin. The investigators hypothesize that oral EHEC may lead to protective immunity against EHEC infection in the gastrointestinal tract. Examination of the immune response to infection with vector strains may aid in elucidating the mechanisms of protective immunity to EHEC and may further the understanding of protective immunity at the mucosal surface. The cloned genes encoding EaeA and EspB from RDEC-1 and StxB1 from EHEC will be placed within the E. coli hemolysis secretion system for export from Vibrio cholerae vaccine strains. The genes will be amplified and cloned into an internal deletion of hlyA such that the gene is fused in frame to the carboxy-terminus of the E. coli hemolysin gene. The resulting constructs will be confirmed by restriction digests and sequence analysis, and placed into V. cholerae strain 0395-NT by electroporation. Localization of EaeA, EspB, and StxB1 in cellular compartments will be performed by ELISA or Western blot analysis using polyclonal anti-EaeA, EspB, and StxB1 antibodies. A rabbit model of Vibrio cholerae colonization will be used to examine the immune response to EaeA, EspB, and StxB1 expressed by the vector strains. Serum and mucosal secretions will be collected from rabbits that have been orally inoculated with the vector strains, and examined for the presence of specific antibody produced in response to immunization. The RDEC-H19A oral challenge model will be used to evaluate protection from the disease induced by immunization with the vaccine strains. Immunized rabbits that develop an immune response to EaeA, EspB, or StxB1 expressed by the vector strains will be orally challenge with the rabbit pathogen RDEC-H19A, and the induction of protective immunity will be evaluated by examining the animals for clinical disease and for histological lesions in the intestine. The proposal is divided into three specific aims: 1) Construct fusions of eaeA, espB, and stxB1 to the E. coli hemolysin secretion signal; and analyze the expression and localization of the hybrid productions. 2) Use the rabbit model to examine the immune responses to EaeA, EspB, and StxB1 expressed by the vector strains. 3) Use the RDEC-H19A rabbit challenge model to assess protection conferred by the immunization.